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Phylogenomics resolves the timing and pattern of insect evolution Bernhard Misof et al. Science 346, 763 (2014); DOI: 10.1126/science.1257570

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Science (print ISSN 0036-8075; online ISSN 1095-9203) is published weekly, except the last week in December, by the American Association for the Advancement of Science, 1200 New York Avenue NW, Washington, DC 20005. Copyright 2014 by the American Association for the Advancement of Science; all rights reserved. The title Science is a registered trademark of AAAS.

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42. Subsequent to the submission of this manuscript, the structure David K. Yeates,4 Kazunori Yoshizawa,38 Qing Zhang,2,3 Rui Zhang,2,3 Wenwei Zhang,3

of a trimeric prefusion HIV-1 Env (43) was determined in complex Yunhui Zhang,3 Jing Zhao,2,3 Chengran Zhou,2,3 Lili Zhou,2,3 Tanja Ziesmann,1

with broadly neutralizing antibodies PGT122 (35) and 35O22 (44). To determine the conformational state these antibodies Shijie Zou,3 Yingrui Li,3 Xun Xu,3 Yong Zhang,2,3 Huanming Yang,3 Jian Wang,3

captured in the crystal lattice, we measured smFRET on labeled Jun Wang,3,39,40,41,42*† Karl M. Kjer,43*† Xin Zhou2,3*† JR-FL virions in the presence of either PGT122 or 35O22 or both. These data indicated that PGT122 strongly stabilized the ground

Insects are the most speciose group of animals, but the phylogenetic relationships of many state. In contrast, 35O22 had little effect on Env conformation. HIV-1 Env complexed with both antibodies exhibited slight ground major lineages remain unresolved. We inferred the phylogeny of insects from 1478 state stabilization. protein-coding genes. Phylogenomic analyses of nucleotide and amino acid sequences,

43. M. Pancera et al., Nature 514, 455–461 (2014). with site-specific nucleotide or domain-specific amino acid substitution models, produced 44. J. Huang et al., Nature 10.1038/nature13601 (2014).

statistically robust and congruent results resolving previously controversial phylogenetic 45. J. S. McLellan et al., Nature 480, 336–343 (2011).

relationships. We dated the origin of insects to the Early Ordovician [~479 million years ago ACKNOWLEDGMENTS (Ma)], of insect flight to the Early Devonian (~406 Ma), of major extant lineages to the We thank J. Jin, L. Agosto, T. Wang, R. B. Altman, and M. R. Wasserman Mississippian (~345 Ma), and the major diversification of holometabolous insects to the for assistance; C. Walsh, J. Binely, A. Trkola, and M. Krystal for reagents; Early Cretaceous. Our phylogenomic study provides a comprehensive reliable scaffold for and members of the Structural Biology Section, Vaccine Research future comparative analyses of evolutionary innovations among insects. Center, for critically reading the manuscript. We thank I. Wilson and J. Sodroski for encouraging us to extend our approach to a R5-tropic Env. The data presented in this paper are tabulated in the nsects (1) were among the first animals to data, however, point to a Cambrian or at least main paper and in the supplementary materials. This work was colonize and exploit terrestrial and freshwa- Early Ordovician origin (4), which implies that supported by NIH grants R21 AI100696 to W.M. and S.C.B; P01 56550 ter ecosystems. They have shaped Earth’s early diversification of insects occurred in marine to W.M., S.C.B., and A.B.S.; and R01 GM098859 to S.C.B.; by the Irvington Fellows Program of the Cancer Research Institute to J.B.M; biota, exhibiting coevolved relationships with or coastal environments. Because of the absence by a fellowship from the China Scholarship Council-Yale World Scholars many groups, from flowering plants to hu- of insect fossils from the Cambrian to the Silurian, Ito X.M.; by grants from the International AIDS Vaccine Initiative’s (IAVI) mans. They were the first to master flight and these conclusions remain highly controversial. Fur-Neutralizing Antibody Consortium to D.R.B., W.C.K., and P.D.K.; and by establish social societies. However, many as- thermore, the phylogenetic relationships among funding from the NIH Intramural Research Program (Vaccine Research Center) to P.D.K. IAVI's work is made possible by generous support pects of insect evolution are still poorly under- major clades of polyneopteran insect orders— from many donors including: the Bill & Melinda Gates Foundation and stood (2). The oldest known fossil insects are including grasshoppers and crickets (Orthoptera), the U.S. Agency for International Development (USAID). This study from the Early Devonian [~412 million years ago cockroaches (Blattodea), and termites (Isoptera)— is made possible by the generous support of the American people (Ma)], which has led to the hypothesis that in- have remained elusive, as has the phylogenetic through USAID. The contents are the responsibility of the authors and do not necessarily reflect the views of USAID or the U.S. government. sects originated in the Late Silurian with the position of the enigmatic Zoraptera. Even the Reagents from the NIH are subject to nonrestrictive material transfer earliest terrestrial ecosystems (3). Molecular closest extant relatives of Holometabola (e.g.,

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beetles, moths and butterflies, flies, sawflies, wasps, metamorphosis), it is important to reliably re- a protein domain–based partitioning scheme to ants, and bees) are unknown. Thus, in order to construct the tempo and mode of insect diversifi- improve the biological realism of the applied understand the origins of physiological and mor- cation. We therefore conducted a phylogenomic evolutionary models) from the representative phological innovations in insects (e.g., wings and study on 1478 single-copy nuclear genes obtained data set (5) (figs. S21, S22, and S23, A and D).

from genomes and transcriptomes representing Trees from both data sets were fully congruent. key taxa from all extant insect orders and other The absence of taxa that cannot be robustly

(ZFMK)/Zentrum für Molekulare Biodiversitätsforschung arthropods (144 taxa) and estimated divergence placed on the tree (rogue taxa) in the amino acid– (ZMB), Bonn, Germany. 2China National GeneBank, BGI- dates with a validated set of 37 fossils (5). sequence data set and the presence of a few Shenzhen, China. 3BGI-Shenzhen, China. 4Australian National

1Zoologisches Forschungsmuseum Alexander Koenig

Phylogenomic analyses of transcriptome and rogue taxa that did not bias tree inference in the Insect Collection, Commonwealth Scientific and Industrial Research Organization (Australia) (CSIRO), National genome sequence data (6) can be compromised nucleotide sequence data set (5) indicated a suf-Research Collections Australia, Canberra, ACT, Australia. by sparsely populated data matrices, gene paral- ficiently representative taxonomic sampling. 5Abteilung Arthropoda, Zoologisches Forschungsmuseum ogy, sequence misalignment, and deviations from To detect confounding signal derived from Alexander Koenig (ZFMK), Bonn, Germany. 6Department of the underlying assumptions of applied evolution- nonrandom data coverage, we randomized ami-Entomology, Rutgers University, New Brunswick, NJ 08854, USA. 7Department of Biological Sciences, Rutgers University, ary models, which may result in biased statistical no acids within taxa, while preserving the dis-Newark, NJ 08854, USA. 8Scientific Computing, Heidelberg confidence in phylogenetic relationships and tem- tribution of data coverage in the representative Institute for Theoretical Studies (HITS), Heidelberg, poral inferences. We addressed these obstacles by data set (5). This approach revealed no evidence Germany. 9Institut für Spezielle Zoologie und removing confounding factors in our analysis (5) of biased node support that could be attributed Evolutionsbiologie mit Phyletischem Museum Jena, FSU Jena, Germany. 10Steinmann-Institut, Bereich Paläontologie, (fig. S2). to nonrandom data coverage (5) (figs. S11 and Universität Bonn, Germany. 112. Zoologische Abteilung We sequenced more than 2.5 gigabases (Gb) of S12 and table S20). Phylogenomic data may (Insekten), Naturhistorisches Museum Wien, Vienna, Austria. cDNA from each of 103 insect species, which violate the assumption of time-reversible evolu-12Department of Integrative Zoology, Universität Wien, represented all extant insect orders (5). Addition- tionary processes, irrespective of what partition Vienna, Austria. 13Institut für Spezifische Prophylaxe und Tropenmedizin, Medizinische Parasitologie, Medizinische ally, we included published transcript sequence scheme one applies, which could lead to in-Universität Wien (MUW), Vienna, Austria. 14Manaaki Whenua data that met our standards (table S2) and offi- correct tree estimates and biased node support. Landcare Research, Auckland, New Zealand. 15Center for cial gene sets of 14 arthropods with sequenced Because sections in the amino acid–sequence Advanced Modeling, Emergency Medicine Department, Johns draft genomes (5), of which 12 served as refer- alignments of the representative data set violat-Hopkins University, Baltimore, MD 21209, USA. 16Biozentrum Grindel und Zoologisches Museum, Universität Hamburg, ences during orthology prediction of transcripts ing these assumptions were present, we tested Hamburg, Germany. 17Evolutionary Morphology Laboratory, (tables S2 and S4). Comparative analysis of the whether the observed compositional heteroge-Graduate School of Science and Engineering, Ehime reference species' official gene sets identified 1478 neity across taxa biased node support but found University, Japan. 18Sugadaira Montane Research Center/ single-copy nuclear genes present in all these no evidence for this (5) (fig. S20). We next dis-Hexapod Comparative Embryology Laboratory, University of Tsukuba, Japan. 19Land and Water Flagship, CSIRO, species (tables S3 and S4). Functional annotation carded data strongly violating the assumption Canberra, ACT, Australia. 20Florida Museum of Natural of these genes revealed that many serve basic of time-reversible evolutionary processes (tables History, University of Florida, Gainesville, FL 32611, USA. cellular functions (tables S14 and S15 and figs. S4 S21 and S22, data files S6 to S8, and figs. S13 to 21Entomology, Staatliches Museum für Naturkunde Stuttgart to S6). A graph-based approach using the best S19). Results from phylogenetic analysis of this (SMNS), Germany. 22Ecology Evolution and Genetics, Research School of Biology, Australian National University, reciprocal genome- and transcriptome-wide hit filtered data set (5) were fully congruent with Canberra, ACT, Australia. 23National Evolutionary Synthesis criterion identified, on average, 98% of these genes those obtained from analyzing the unfiltered Center, Durham, NC 27705, USA. 24Department of Biological in the 103 de novo sequenced transcriptomes, but representative data set. The nucleotide sequence Sciences, Macquarie University, Sydney, Australia. only 79% and 62% in the previously published data of the representative data set containing 25Department für Botanik und Biodiversitätsforschung, Universität Wien, Vienna, Austria. 26Natural History Museum transcriptomes of in- and out-group taxa, respec- also first and third codon positions strongly violated of Crete, University of Crete, Post Office Box 2208, tively (tables S12 and S13). the assumption of time-reversible evolutionary Gr-71409, Iraklio, and Biology Department, University of After transcripts had been assigned and aligned processes, but still supported largely congruent Crete, Iraklio, Crete, Greece. 27Department of Biological to the 1478 single-copy nuclear-encoded genes, topologies (fig. S23, B to D). In summary, our Sciences and Feinstone Center for Genomic Research, University of Memphis, Memphis, TN 38152, USA. 28Centro we checked for highly divergent, putatively phylogenetic inferences are unlikely to be biased Universitario de Ciencias Biólogicas y Agropecuarias, Centro misaligned transcripts. Of the 196,027 aligned by any of the above-mentioned confounding factors. de Estudios en Zoología, Universidad de Guadalajara, transcripts, 2033 (1%) were classified as highly Our phylogenomic study suggests an EarlyZapopan, Jalisco, México. 29Leibniz Supercomputing Centre divergent. Of these, 716 were satisfactorily re- Ordovician origin of insects (Hexapoda) at ~479 Ma of the Bavarian Academy of Sciences and Humanities, Garching, Germany. 30Institute of Evolutionary Biology and aligned with an automated refinement. How- [confidence interval (CI), 509 to 452 Ma] and a Ecology, Zoology and Evolutionary Biology, University of ever, alignments of 1317 transcripts could not be radiation of ectognathous insects in the Early Bonn, Bonn, Germany. 31Department of Life Sciences, The improved, and these transcripts were excluded Silurian ~441 Ma (CI 465 to 421 Ma) (Figs. 1 and 2). Natural History Museum London, London, UK. 32Abteilung from our analyses (supplementary data file S5, These estimates imply that insects colonizedEntomologie, Biozentrum Grindel und Zoologisches Museum, Universität Hamburg, Hamburg, Germany. 33Fakultät für http://dx.doi.org/10.5061/dryad.3c0f1). land at roughly the same time as plants (8), in Informatik, Karlsruher Institut für Technologie, Karlsruhe, Nonrandom distribution of missing data among agreement with divergence date estimates on Germany. 34California Academy of Sciences, San Francisco, taxa can inflate statistical support for incorrect the basis of other molecular data (4).CA 94118, USA. 35Department of Entomology, College of tree topologies (7). Because we detected a non- The early diversification pattern of insects has Natural Resources and Environment, South China Agricultural University, China. 36Yokosuka City Museum, random distribution of missing data, we only remained unclear (2, 7, 9). We received support Yokosuka, Kanagawa, Japan. 37Department of Entomology, considered data blocks if they contained infor- for a monophyly of insects, including Collembola North Carolina State University, Raleigh, NC 27695, USA. mation from at least one representative of each and Protura as closest relatives (10), and Diplura 38Systematic Entomology, Hokkaido University, Sapporo, of the 39 predefined taxonomic groups of un- as closest extant relatives of bristletails (Archae-Japan. 39Department of Biology, University of Copenhagen, Copenhagen, Denmark. 40Princess Al Jawhara Center of disputed monophyly (table S6). In this represent- ognatha), silverfish (Zygentoma), and winged in-Excellence in the Research of Hereditary Disorders, King ative data set, the extent of missing data was still sects (Pterygota) (Fig. 1). Furthermore, our analyses Abdulaziz University, Jeddah, Saudi Arabia. 41Macau between 5 and 97.7% in pairwise sequence com- corroborate Remipedia, cave-dwelling crustaceans, University of Science and Technology, Avenida Wai Long, parisons, with high percentages primarily because as the closest extant relatives of insects (11, 12).Taipa, Macau, China. 42Department of Medicine, University of Hong Kong, Hong Kong. 43Department of Ecology, Evolution, of the data scarcity in some previously published A close phylogenetic relationship of bristletails and Natural Resources, Rutgers University, New Brunswick, out-group taxa (table S19 and figs. S7 to S10). to a clade uniting silverfish and winged insects NJ 08854, USA. We inferred maximum-likelihood phylogenetic (Dicondylia) is generally accepted. However, the *Major contributors. trees (Fig. 1) with both nucleotide (second-codon monophyly of silverfish has been questioned,†Corresponding author. E-mail: xinzhou@genomics.cn (X.Z.), b.misof.zfmk@uni-bonn.de (B.M.), kjer@aesop.rutgers.edu positions only and applying a site-specific rate with the relict Tricholepidion gertschi considered (K.M.K), wangj@genomics.cn (J.W.) model) and amino acid–sequence data (applying more distantly related to winged insects than

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Fig. 1. Dated phylogenetic tree of insect relationships.The tree was inferred through a maximum-likelihood analysis of 413,459 amino acid sites divided into 479 metapartitions. Branch lengths were optimized and node ages estimated from 1,050,000 trees sampled from trees separately generated for 105 partitions that included all taxa (5). All nodes up to orders are labeled with numbers (gray circles). Colored circles indicate bootstrap support (5) (left key). The time line at the bottom of the tree relates the geological origin of insect clades to major geological and biological events. CONDYLO, Condylognatha; PAL, Palaeoptera.

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Fig. 2. Sorted ordinal and interordinal node age estimates. For each labeled of age estimates separately derived from each metapartition. Within the bean node (numbers on the left and right of the figure correspond to the node labels plot (gray scale), blue bars indicate the distribution of median age estimates, in the tree of Fig. 1), the median (red bar), and the range of the upper and lower large blue bars indicate the inferred median of medians. All node age estimates confidence interval (black rectangle) of age estimates are illustrated. These refer to the estimated common origin of included species. Stem-lineage repre-medians and upper and lower confidence intervals are derived from uniformly sentatives can, of course, be older. The maximum root age of the tree was set sampled trees over all 105 metapartitions (5). Additionally, we present medians to 580 Ma to coincide with the oldest Ediacaran fossils (5).

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other silverfish (13). We find that silverfish are monophyletic, consistent with recently published morphological studies (14), and estimate that Tricholepidion diverged from other silverfish in the Late Triassic (~214 Ma) (Figs. 1 and 2). This result implies parallel and independent loss of the ligamentous head endoskeleton, abdominal styli, and coxal vesicles in winged insects and silverfish (5). The diversification of insects is undoubtedly

related to the evolution of flight. Fossil winged insects exist from the Late Mississippian (~324 Ma) (15), which implies a pre-Carboniferous origin of insect flight. The description of †Rhyniognatha (~412 Ma) from a mandible, potentially indicative of a winged insect, suggested an Early Devonian to Late Silurian origin of winged insects (3). Our results corroborate an origin of winged insect lineages during this time period (16) (Figs. 1 and 2), which implies that the ability to fly emerged after the establishment of complex terrestrial ecosystems. Ephemeroptera and Odonata are, according to

our analyses, derived from a common ancestor. However, node support is low for Palaeoptera (Ephemeroptera + Odonata) and for a sister group relationship of Palaeoptera to modern winged in-sects (Neoptera), which indicates that additional evidence, including extensive taxon sampling and the analysis of genomic meta-characters (17), will be necessary to corroborate these relationships. We find strong support for the monophyly of

Polyneoptera, a group that comprises earwigs, stone-flies, grasshoppers, crickets, katydids (Orthoptera), Embioptera, Phasmatodea, Mantophasmatodea, Grylloblattodea, cockroaches, mantids, termites, and Zoraptera (18–20). We estimated the origin of the polyneopteran lineages at ~302 Ma (CI 377 to 231 Ma) in the Pennsylvanian (Figs. 1 and 2), con-sistent with the idea that at least part of the rich Carboniferous neopteran insect fauna was of poly-neopteran origin. Finally, our analyses suggest that the major diversity within living cockroaches, man-tids, termites, and stick insects evolved after the Permian mass extinction. Given that the oldest known fossil hemipterans

date to the Middle Pennsylvanian (~310 Ma) (21), it had been thought that the stylet marks on liv-erworts from the Late Devonian (~380 Ma) (22) could not have been of hemipteran origin. Our study indicates that true bugs (Hemiptera) and their sister lineage, thrips (Thysanoptera), all of which possess piercing-sucking mouthparts, orig-

inated ~373 Ma (CI 401 to 346 Ma), which gives support to the possibility of a hemipteroid origin of Early Paleozoic stylet marks. True bugs, thrips, bark lice (Psocoptera), and

true lice (Phthiraptera) (together called Acercaria) were thought to be the closest extant relatives of Holometabola (Acercaria + Holometabola = Eumetabola) (10). However, convincing morpho-logical features and fossil intermediates support-ing a monophyly of Acercaria are lacking (13). We recovered bark and true lice (Psocodea) as likely closest extant relatives of Holometabola (5), which suggests that both groups started to diverge in the Devonian-Mississippian ~362 Ma (CI 390 to 334 Ma) (Figs. 1 and 2). However, this result did not receive support in all statistical tests and, therefore, should be further investigated in future studies that embrace additional types of characters (17). We estimated that the radiation of parasitic

lice occurred ~53 Ma (CI 67 to 46 Ma), which implies that they diversified well after the emer-gence of their avian and mammalian hosts in the Late Cretaceous–Early Eocene and contradicts the hypothesis that parasitic lice originated on fea-thered theropod dinosaurs ~130 Ma (23). Within Holometabola, our study recovered

phylogenetic relationships fully congruent with those suggested in recent studies (2, 24, 25). Although we estimated the origin of stem lineages of many holometabolous insect orders in the Late Carboniferous, we dated the spectacular diver-sifications within Hymenoptera, Diptera, and Lepidoptera to the Early Cretaceous, contem-porary with the radiation of flowering plants (21, 26). The almost linear increase in interordinal insect diversity suggests that the process of di-versification of extant insects may not have been severely affected by the Permian and Cretaceous biodiversity crises (Fig. 2). With this study, we have provided a robust

phylogenetic backbone tree and reliable time estimates of insect evolution. These data and analyses establish a framework for future com-parative analyses on insects, their genomes, and their morphology.

REFERENCES AND NOTES

1. The term “insects” is used here in a broad sense and synonymous to Hexapoda (including the ancestrally wingless Protura, Collembola, and Diplura).

2. M. D. Trautwein, B. M. Wiegmann, R. Beutel, K. M. Kjer, D. K. Yeates, Annu. Rev. Entomol. 57, 449–468 (2012).

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ACKNOWLEDGMENTS

The data reported in this paper are tabulated in the supplementary materials and archived at National Center for Biotechnology Information, NIH, under the Umbrella BioProject ID PRJNA183205 (“The 1KITE project: evolution of insects”). Supplementary files are archived at the Dryad Digital Repository http://dx.doi.org/ 10.5061/dryad.3c0f1. Funding support: China National GeneBank and BGI-Shenzhen, China; German Research Foundation (NI 1387/ 1-1; MI 649/6, MI 649/10, RE 345/1-2, BE1789/8-1, BE 1789/10-1, STA 860/4, Heisenberg grant WA 1496/8-1); Austria Science Fund FWF; NSF (DEB 0816865); Ministry of Education, Culture, Sports, Science and Technology of Japan Grant-in-Aid for Young Scientists (B 22770090); Japan Society for the Promotion of Science (P14071); Deutsches Elektronen-Synchrotron (I-20120065); Paul Scherrer Institute (20110069); Schlinger Endowment to CSIRO Ecosystem Sciences; Heidelberg Institute for Theoretical Studies; University of Memphis-FedEx Institute of Technology; and Rutgers University. The authors declare no conflicts of interest.

SUPPLEMENTARY MATERIALS

www.sciencemag.org/content/346/6210/763/suppl/DC1 Materials and Methods Supplementary Text Figs. S1 to S27 Tables S1 to S26 Data File Captions S1 to S14 References (27–187)

17 June 2014; accepted 23 September 2014 10.1126/science.1257570

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